Materials and methods for treating or preventing oxalate-related disease

ABSTRACT

This invention provides materials and procedures for the delivery of selected strains of bacteria and/or oxalate-degrading enzymes to the intestinal tracts of persons and/or domestic or zoological animals who are at increased risk for oxalate related disease because they have lost, or have inadequate concentrations of these bacteria. The administration of these bacteria and/or the relevant enzyme removes oxalate from the intestinal tract and thus reduces the amount of oxalate available for absorption and reduces the risk for oxalate related disease.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.09/083,362, filed May 22, 1998 now U.S. Pat. No. 6,200,562 which claimsthe benefit of U.S. Provisional Application No. 60/047,473, filed May23, 1997. This application also claims the benefit of U.S. ProvisionalApplication No. 60/150,259, filed Aug. 23, 1999.

BACKGROUND OF THE INVENTION

Kidney-urinary tract stone disease (urolithiasis)is a major healthproblem throughout the world. Most of the stones associated withurolithiasis are composed of calcium oxalate alone or calcium oxalateplus calcium phosphate. Other disease states have also been associatedwith excess oxalate. These include, vulvodynia, oxalosis associated withend-stage renal disease, cardiac conductance disorders, Crohn's disease,and other enteric disease states.

Oxalic acid (and/or its salt-oxalate) is found in a wide diversity offoods, and is therefore, a component of many constituents in human andanimal diets. Increased oxalate absorption may occur after foodscontaining elevated amounts of oxalic acid are eaten. Foods such asspinach and rhubarb are well known to contain high amounts of oxalate,but a multitude of other foods and beverages also contain oxalate.Because oxalate is found in such a wide variety of foods, diets that arelow in oxalate and which are also palatable are hard to formulate. Inaddition, compliance with a low oxalate diet is often problematic.

Endogenous oxalate is also produced metabolically by normal tissueenzymes. Oxalate (dietary oxalate that is absorbed as well as oxalatethat is produced metabolically) is not further metabolized by tissueenzymes and must therefore be excreted. This excretion occurs mainly viathe kidneys. The concentration of oxalate in kidney fluids is critical,with increased oxalate concentrations causing increased risk for theformation of calcium oxalate crystals and thus the subsequent formationof kidney stones.

The risk for formation of kidney stones revolves around a number offactors that are not yet completely understood. Kidney-urinarytractstone disease occurs in as much as 12% of the population in Westerncountries and about 70% of these stones are composed of calcium oxalateor of calcium oxalate plus calcium phosphate. Some individuals (e.g.,patients with intestinal disease such as Crohn's disease, inflammatorybowel disease, or steatorrhea and also patients that have undergonejejunoileal bypass surgery) absorb more of the oxalate in their dietsthan do others. For these individuals, the incidence of oxalateurolithiasis increases markedly. The increased disease incidence is dueto increased levels of oxalate in kidneys and urine, and this, the mostcommon hyperoxaluric syndrome in man, is known as enteric hyperoxaluria.Oxalate is also a problem in patients with end-stage renal disease andthere is recent evidence (Solomons, C. C., M. H. Melmed, S. M. Heitler[1991] “Calcium citrate for vulvar vestibulitis” Journal of ReproductiveMedicine 36:879-882) that elevated urinary oxalate is also involved invulvar vestibulitis (vulvodynia).

Bacteria that degrade oxalate have been isolated from human feces(Allison, M. J., H. M. Cook, D. B. Milne, S. Gallagher, R. V. Clayman[1986] “Oxalate degradation by gastrointestinal bacteria from humans” JNutr. 116:455-460). These bacteria were found to be similar tooxalate-degradingbacteria that had been isolated from the intestinalcontents of a number of species of animals (Dawson, K. A., M. J.Allison, P. A. Hartman [1980] “Isolation and some characteristics ofanaerobic oxalate-degradingbacteria the rumen” Appl. Environ. Microbiol.40:833-839; Allison, M. J., H. M. Cook [1981] “Oxalate degradation bymicrobes of the large bowel of herbivores: the effect of dietaryoxalate” Science 212:675-676; Daniel, S. L., P. A. Hartman, M. J.Allison [1987] “Microbial degradation of oxalate in the gastrointestinaltracts of rats” Appl. Environ. Microbiol. 53:1793-1797). These bacteriaare different from any previously described organism and have been givenboth a new species and a new genus name (Allison, M. J., K. A. Dawson,W. R. Mayberry, J. G. Foss [1985] “Oxalabacterformigenes gen. nov., sp.nov.:oxalate-degradinganaerobes that inhabit the gastrointestinal tract”Arch. Microbiol. 141:1-7).

Not all humans carry populations of O. formigenes in their intestinaltracts (Allison, M. J., S. L. Daniel, N. A. Comick [1995]“Oxalate-degrading bacteria” In Khan, S. R. (ed.), Calcium Oxalate inBiological Systems CRC Press; Doane, L. T., M. Liebman, D. R. Caldwell[1989] “Microbial oxalate degradation: effects on oxalate and calciumbalance in humans” Nutrition Research 9:957-964). There are lowconcentrations or a complete lack of oxalate degrading bacteria in thefecal samples of persons who have had jejunoileal bypass surgery(Allison et al. [1986] “Oxalate degradation by gastrointestinal bacteriafrom humans” J. Nutr. 116:455-460). Also, certain humans and animals maymaintain colonies of O. formigenes but nevertheless have excess levelsof oxalate for reasons which are not clearly understood.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to materials and methods which reduce therisk for developing oxalate-related disorders by reducing the amount ofoxalate in the intestinal tract. This reduction in the intestinal tractleads to a reduction in systemic oxalate levels thereby promoting goodhealth.

In one embodiment of the subject invention, a reduction in oxalateabsorption is achieved by supplying oxalate-degrading bacteria to theintestinal tract. In a preferred embodiment, these bacteria areOxalobacter formigenes. These bacteria use oxalate as a growthsubstrate. This utilization reduces the concentration of soluble oxalatein the intestine and, thus, the amount of oxalate available forabsorption. A reduction of oxalate in the intestinal tract can also leadto removal of oxalate from the circulatory system.

In a specific embodiment, the subject invention provides materials andprocedures for the delivery of O. formigenes to the intestinal tracts ofpersons who are at increased risk for oxalate-related disease. Thesebacteria and their progeny replicate in the intestine and remove oxalatefrom the intestinal tract, thereby reducing the amount of oxalateavailable for absorption (and/or causing oxalate excretion from theblood into the intestine) and thus reducing the risk for oxalate relateddisease.

In accordance with the teaching of the subject invention,oxalate-degradingmicrobes other than O. formigenes which utilize oxalateas a substrate can also be used to achieve therapeutic oxalatedegradation thereby reducing the risk of urolithiasis and otheroxalate-related disorders. Such other microbes may be, for example,bacteria such as clostridia or pseudomonads.

In one embodiment of the subject invention, the microbes which are usedto degrade oxalate naturally produce enzymes which confer upon thesemicrobes the ability to degrade oxalate. In an alternative embodiment,microbes may be transformed with polynucleotide sequences which conferupon the transformed microbes the ability to degrade oxalate.Specifically, the enzymes formyl—CoA transferase and oxalyl—CoAdecarboxylase have been identified as playing a central role in oxalatedegradation. In one embodiment of the subject invention, an appropriatehost can be transformed with heterologous DNA encoding these enzymeactivities thereby conferring upon the transformed host the ability toaugment oxalate degradation. The host may be, for example, a microbewhich is particularly well adapted for oral administration and/orcolonizing the intestines. Alternatively, the host may be a plant which,once transformed, will produce the desired enzyme activities therebymaking these activities available in the intestine when the plantmaterial is consumed.

A further embodiment of the subject invention provides plantstransformed with oxalate-degrading enzymes wherein these plants haveenhanced resistance to fungi which require oxalate for theirpathogenesis of plants or which produce oxalic acid as a mechanisms fortheir pathogenesis of plants.

In a further embodiment of the subject invention, a reduction in oxalatelevels is achieved by administering enzymes which act to degradeoxalate. These enzymes may be isolated and purified or they may beadministered as a cell lysate. The cell lysate may be, for example, O.formigenes. In a specific embodiment, the enzymes which are administeredare formyl-CoA transferase and oxalyl-CoA decarboxylase. In a preferredembodiment, additional factors which improve enzyme activity can beadministered. These additional factors may be, for example, oxalyl CoA,MgCl₂, and TPP (thiamine diphosphate, an active form of vitamin B₁).

Thus, in one embodiment of the subject invention, a reduction in oxalatelevels is achieved by administering oxalate-degrading enzymes producedby a recombinant microbe, such as Escherichia coli, which has beentransformed to express oxalate-degradingenzymes. The recombinant hostmay be administered in either a viable or non-viable form. A furtheraspect of the subject invention pertains to pharmaceutical compositionsand/or nutritional supplements for oral administration. Thesecompositions release the oxalate degrading microbes, or oxalatedegrading enzymes, in the intestines of humans or animals. Preferablythe microorganisms and/or enzymes are encapsulated in a dose deliverysystem that decreases the probability of release of the materials in thehuman or animal stomach but increases the probability of release in theintestines. The microorganisms and/or enzymes also may be administeredas a constituent of foods, such as milk, meats, and yogurt.

In a further embodiment of the subject invention, a reduction in oxalateabsorption is achieved in domesticated, agricultural, or zool-maintainedanimals deficient in oxalate-degrading bacteria by administeringoxalate-degrading microorganisms, plants, and/or enzymes.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a shows the results of a study evaluating the conversion oflabeled carbon (C-14) from dietary oxalate into carbon dioxide whenOxalobacter formigenes cells are fed to rats on a high calcium diet.

FIG. 1b shows the results of a study evaluating the conversion oflabeled carbon (C-14) from dietary oxalate into carbon dioxide whenOxalobacter formigenes cells are fed to rats on a low calcium diet.

FIG. 2a shows the results of a further study evaluating the fate ofdietary oxalate when Oxalobacter formigenes cells are included in thediet.

FIG. 2b shows the results of a further study evaluating the fate ofdietary oxalate when Oxalobacter formigenes cells are included in thediet.

FIG. 2c shows the results of a further study evaluating the fate ofdietary oxalate when Oxalobacter formigenes cells are included in thediet.

FIGS. 3a-c show the reduction in urinary excretion of oxalate in pigswhich are fed oxalobacter.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention pertains to the introduction of oxalate-degradingbacteria and/or enzymes into a human or animal intestinal tract wherethe activity of these materials reduces the amount and/or concentrationof oxalate present thereby reducing the risk of disease due to oxalate.

In a specific embodiment, the subject invention pertains to thepreparation and administration of cells of oxalate-degrading bacteria ofthe species, Oxalobacter formigenes, to the human or animal intestinaltract where the activity of the microbes reduces the amount of oxalatepresent in the intestine thereby causing a reduction of concentrationsof oxalate in the kidneys and in other cellular fluids. The introducedcells degrade oxalate and replicate in the intestinal habitat so thatprogeny of the initial cells colonize the intestine and continue toremove oxalate. This activity reduces the risk for formation of kidneystones as well as other disease complications caused by oxalic acid. Ina preferred embodiment for human use, the specific strains of O.formigenes used are strains isolated from human intestinal samples. Thestrains are thus part of the normal human intestinal bacterial flora.However, since they are not present in all persons, or are present ininsufficient numbers, the introduction of these organisms corrects adeficiency that exists in some humans.

Enrichment of the contents of the intestines with one or more species ofoxalate-degrading bacteria causes a reduction of oxalate in theintestinal contents. Some of the bacteria carry out oxalate degradationat or near the site of absorption. The activity of the bacteriadecreases the level of absorption of dietary oxalate. A reduction inoxalate concentration in the intestines can also lead to a removal ofoxalate from cells and the general circulation. More specifically, areduction of oxalate concentration in the intestines can also lead toenhanced secretion of oxalate into the intestine from the blood and thusreduce the amount of oxalate that needs to be excreted in urine. Thus,the methods of the subject invention can be used to treat primaryhyperoxaluria in addition to treatment of dietary hyperoxaluria. Thematerials and methods of the subject invention are particularlyadvantageous in the promotion of healthy oxalate levels in humans andanimals.

Pharmaceutical and nutriceutical compositions for the introduction ofoxalate degrading bacteria and/or enzymes into the intestine includebacteria and/or enzymes that have been lyophilized or frozen in liquidor paste form and encapsulated in a gel capsule or other entericprotection. The gel cap material is preferably a polymeric materialwhich forms a delivery pill or capsule that is resistant to degradationby the gastric acidity and enzymes of the stomach but is degraded withconcomitant release of oxalate-degrading materials by the higher pH andbile acid contents in the intestine. The released material then convertsoxalate present in the intestine to harmless products. Pharmaceutical ornutriceutical carriers also can be combined with the bacteria orenzymes. These would include, for example, saline-phosphate buffer.

Bacteria and/or enzymes to be administered can be delivered as capsulesor microcapsules designed to protect the material from adverse effectsof acid stomach. One or more of several enteric protective coatingmethods can be used. Descriptions of such enteric coatings include theuse of cellulose acetate phthalate (CAP) (Yacobi, A., E. H. Walega,1988, Oral sustained release formulations: Dosing and evaluation,Pergammon Press). Other descriptions of encapsulation technology includeU.S. Pat. No. 5,286,495, which is incorporated herein by reference. Thecompositions of the subject invention can also be formulated assuppositories.

Other methods of administration of these microorganisms and/or enzymesto the intestines include adding the material directly to food sources.The bacteria may be added as freshly harvested cells, freeze driedcells, or otherwise protected cells. Foods may be supplemented withoxalate degrading organisms without affecting their taste or appearance.These foods may be, for example, yogurt, milk, peanut butter orchocolate. Upon ingestion, when the food products are being digested andabsorbed by the intestines, the microorganisms and/or enzymes degradeoxalate present in the intestines thus reducing absorption of oxalateinto the blood stream.

As noted above, a variety of foods can be supplemented with oxalatedegrading microorganisms. As an initial step, the microbes can be grownin media and separated from the media by, for example, centrifugation.Traditional yogurt cultures obtained from a commercial dairy can bemixed with the oxalate degrading microbial culture. This mixture ofcultures then can be added to the basic dairy yogurt premix withoutadversely affecting taste or consistency. The yogurt can then beproduced and packaged using traditional commercial procedures. Inanother example, the oxalate degrading bacteria can be added to alreadyproduced yogurts.

Another example is to add the microbes to milk after it has beenhomogenized and sterilized. Such a method is currently used in the diaryindustry for adding Lactobacillus acidophilus organisms to milk. Anyfood source containing bacteria can be used by supplementing withoxalate-degrading bacteria. These food products include cheese or meatproducts that have desirable microorganisms added during processing.

In yet a further embodiment, the subject invention provides a novelenzyme delivery system. This system comprises a plant which has beentransformed with heterologous polynucleotide(s) to expressoxalate-degradingenzymes. The enzyme-expressing transgenic plant may beadministered to patients as a constituent of a salad, for example.Further, the enzyme-expressing plant may be administered to animals as aconstituent of feed, for example, or grown in grazing pasture. Theanimals to which these products may be fed include, for example, cattle,pigs, dogs and cats.

Thus, as an alternative method of administration to the intestine,plants are genetically engineered to express oxalate-degrading enzymes.These transgenic plants are added to the diet, with the activity of theenzymes causing a decrease in the presence of oxalate. DNA sequencesencoding these enzymes are known to those skilled in the art and aredescribed in, for example, WO 98/16632.

In addition to plants which can be used as a dietary component topromote healthy oxalate levels in humans or animals, the subjectinvention provides plants with enhanced resistance to microbialinfections. Specifically, the transformed plants of the subjectinvention are protected against microbes which require or use thepresence of oxalate for plant pathogenicity. The plants of the subjectinvention, which are transformed to express oxalate-degrading enzymesare protected against, for example, certain fungi which need oxalate forpathogenicity. The genes encoding the enzymes can be modified to enhanceexpression and/or stability in plants. Also, the expression may be underthe control of promoters which direct expression in particular tissues.

In one embodiment, the strains of bacteria (O. formigenes) usedaccording to the subject invention are pure cultures that are isolatedfrom anaerobic cultures that have been inoculated with dilutions ofintestinal contents from normal humans or, for use with animals, fromnormal animals. A special calcium oxalate containing medium that allowsdetection of oxalate degrading colonies can be used. In one embodiment,the purity of each strain can be assured through the use of at least twosubsequent repetitive cloning steps.

Strains of O. formigenes useful according to the subject invention havebeen characterized based upon several tests, these include: patterns ofcellular fatty acids, patterns of cellular proteins, DNA and RNA(Jensen, N. S., M. J. Allison (1995) “Studies on the diversity amonganaerobic oxalate degrading bacteria now in the species Oxalobacterformigenes” Abstr. to the General Meeting of the Amer. Soc. Microbiol.,1-29), and responses to oligonucleotide probes (Sidhu et al. 1996). Twogroups of these bacteria (Groups I and II, both existing within thepresent description of the species) have been described. Strains usedhave been selected based upon oxalate degrading capacity, and evidenceof the ability to colonize the human intestinal tract. Strains selectedinclude representatives of both Groups I and II of the species.

One embodiment of the present invention involves procedures forselection, preparation and administration of the appropriateoxalate-degrading bacteria to a diversity of subjects. Prominently, butnot exclusively, these are persons or animals which do not harbor thesebacteria in their intestines. These non-colonized or weakly-colonizedpersons or animals are identified using tests that allow for rapid anddefinitive detecting of O. formigenes even when the organisms are atrelatively low concentrations in mixed bacterial populations such as arefound in intestinal contents. The methods of the subject invention canalso be used to treat individuals or animals whose oxalate-degradingbacteria have been depleted due to, for example, antibiotic treatment orin post-operative situations. The methods of the subject invention canalso be used to treat individuals or animals who have colonies ofoxalate-degradingbacteria but who still have unhealthy levels of oxalatedue to, for example, oxalate susceptibility and/or excessive productionof endogenous oxalate.

Bacteria which can be used according to the subject invention can beidentified by at least two methods:

1) Oligonucleotide probes specific for these bacteria can be used;and/or

2) A culture test wherein an anaerobic medium with 10 mM oxalate isinoculated and after incubation at 37° C. for 1 to 7 days, the loss ofoxalate is determined.

Pure cultures of O. formigenes strains can be grown in large fermenterbatch cultures and cells can be harvested using techniques known tothose skilled in the art. Cells from a selected single strain ormixtures of known strains can be treated as needed (e.g., freeze driedwith trehalose or glycerol) to preserve viability and are then placed incapsules designed to protect the cells through their passage through theacid stomach (enteric coated capsules).

Cells are ingested in quantities and at intervals determined by theneeds of individuals. In some cases a single, or periodic, use may beall that is needed and in other cases regular ingestion (e.g., withmeals) may be needed.

The invention further pertains to administration to the human or animalintestinal tract of oxalate-degrading products or enzymes prepared fromO. formigenes cells. In one embodiment, oxalate degrading enzymes can bepurified and prepared as a pharmaceutical or nutriceutical compositionfor oral consumption. In a preferred embodiment, these enzymes areproduced recombinantly. DNA sequences encoding these enzymes are knownto those skilled in the art and are described in, for example, WO98/16632. These sequences, or other sequences encoding oxalate-degradingproteins, can be expressed in a suitable host. The host may be, forexample, E. coli or Lactobacillus. The transformed host would includedappropriate regulatory and transporter signals. The expressed proteinmay be isolated, purified and administered as described herein.Alternatively, the recombinant host expressing the desiredoxalate-degrading proteins may be administered. The recombinant host maybe administered in either a viable or non-viable form. In anotherpreferred embodiment, the enzymes are coated or otherwise formulated ormodified to protect the enzymes so that they are not inactivated in thestomach, and are available to exert their oxalate-degrading activity inthe small intestine. Examples of such formulations are known to thoseskilled in the art and are described in, for example, U.S. Pat. No.5,286,495.

The use of O. formigenes is particularly advantageous because it is ananaerobe that does not grow in aerobic tissue environments and does notproduce any compounds which are toxic to humans or animals. As analternative to either O. formigenes or a recombinant host, otheroxalate-degrading bacteria may be used, such as Clostridium orPseudomonas. Oxalate-degrading enzymes prepared from such alternativebacteria may be administered or the entire microbe may be administered.

In addition, all aforementioned embodiments are applicable todomesticated, agricultural, or zoo-maintained animals suffering fromdeficient numbers of oxalate-degrading bacteria, as well as to humans.For example, oxalate-degrading enzymes and/or microbes may beadministered to house pets such as dogs, cats, rabbits, ferrets, guineapigs, hamsters and gerbils, as well as to agricultural animals, such ashorses, sheep, cows and pigs, or wild animals maintained for breedingpurposes such as river otters.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting.

EXAMPLE 1

Treatment of High Risk Patients

Enteric coated O. formigenes cells can be ingested by patientpopulations at high risk for oxalate related disease. These include:

1. Persons that have a history of urolithiasis with multiple episodes ofidiopathic stone disease.

2. Persons at risk for urolithiasis with high urinary oxalate due toenteric disease (enteric-hyperoxaluria).

3. Persons with high serum oxalate levels due to end stage renaldisease.

4. Persons with vulvar vestibultitis.

5. Persons that have diets with high levels of oxalate such as found incertain areas and seasons in India and in Saudi Arabia. This would alsoinclude individuals who happen to prefer foods such as spinach which arehigh in oxalate.

6. Persons who produce too much endogenous oxalate due to, for example,a genetic defect.

EXAMPLE 2

Treatment of Low Risk Patients

Enteric protected O. formigenes cells can also be ingested byindividuals in populations at lower risk for oxalate related disease.These include:

1. Persons that have lost populations of normal oxalate degradingbacteria due to: treatments with oral antibiotics or bouts of diarrhealdisease.

2. Infants can be inoculated so that a normal protective population ofOxalabacter will be more easily established than is the case later inlife when competitive exclusion principles operate.

EXAMPLE 3

Use of Oxalate Degrading Enzymes From Oxalobacter formigenes to ControlHyperoxaluria

A study was conducted to evaluate the efficacy of oxalate degradingenzymes from Oxalobacter formigenes for the control of hyperoxaluria.

Animals Used: Male Sprague Dawley Rats: BW 250-300 g

Diets Used: Normal Diet (N.D.): Harlan Teklad TD 89222; 0.5% Ca, 0.4%P

Drug Used: Lyophilized mixture of Oxalobacter formigenes lysate (sourceof enzymes) with Oxalyl CoA, MgCl₂ and TPP.

Drug Delivery System (Capsules): Size 9 capsules for preclinical ratstudies (Capsu-Gel). Enteric Coating EudragitL-100-55 (Hulls America,Inc.). Basal 24 hr urine collection. Fecal analysis for Oxalobacterformigenes—rats were not colonized with Oxalobacter formigenes.

Experimental Protocol:

A. Long-term Studies

Animal Protocol

Group I (n=4): Fed oxalate diet with lysate. Rats were given twocapsules everyday at 4:00 p.m. and oxalate diet overnight. Diet wasremoved during the day (8:00 a.m. to 4:00 p.m.)

Group II (n=4): Fed oxalate diet as described for Group I (HyperoxaluricControls).

24 hr urine samples were collected on Day 7 and Day 9 of the abovetreatment.

Data on the mean urinary oxalate concentration for the two groups ofrats shown above indicated that feeding of Oxalobacter lysate loweredthe urinary oxalate concentration in Group I rats as compared to thehyperoxaluric controls (Group II). The enzymes can not be active for along duration in the gastrointestinal tract; therefore, short-termstudies were performed as described below.

B. Short-term Studies

Animal Protocol

Group I (n=4): Fed 1 capsule at 8:00 a.m.; oxalate diet for two hours(rats were fasted overnight so that they eat well during this period)and 1 capsule at 10:00 a.m.

Group II (n=4): Oxalate diet for two hours as for Group I.

Urine was collected from all the animals for the next five-hour periodand analyzed for oxalate concentration. This was performed on days 11,12 and 15 of this study.

The results of this study show that feeding the Oxalobacter lysateproduces a significant decrease in urinary oxalate levels in a 5 hourperiod after oxalate and drug administration in Group I rats as comparedto the hyperoxaluric control group (Group II). At this point a crossoverstudy between the two groups of rats was performed.

C. Cross-Over Studies

Animal Protocol

Group I: Fed oxalate diet twice a day at 8:00-10:00 a.m. and 3:00p.m-5:00 p.m.

Group II: Fed 1 capsule twice a day before feeding the oxalate diet asfor Group I.

Short-term studies for the effect of oxalobacter lysate feeding onurinary oxalate levels were performed as described in Section-B above onday-2 and day-5 after the cross-over.

Crossover studies show that previously hyperoxaluric Group II rats,which are now being fed the Oxalobacter lysate, show a decline inurinary oxalate levels. In contrast the Group-I rats revert tohyperoxaluria upon withdrawal of the drug.

EXAMPLE 4

Treatment with Oxalobacter formigenes Cells to Rats

A study was conducted to evaluate the fate of dietary oxalate whenOxalobacter formigenes cells are included in the diet.

Methods

Male Wistar rats were fed a normal calcium (1%), high oxalate (0.5%)diet, or a low calcium (0.02%), high oxalate diet (0.5%) diet during twoseparate experiments. ¹⁴C-oxalate (2.0 uCi) was given on day 1 and againon day 7 of the study. Oxalobacter formigenes cells (380 mg/d) wereadministered in rat drinking water on days 5-11. The fate of ¹⁴C fromoxalate was measured based on analysis of ¹⁴C in feces, urine andexpired air. The rats served as self controls and measurements duringthe control period (before Oxalobacter cells were fed) were made duringdays 1-4; during the experimental period (when bacterial cells were fed)measurements were made on days 7-11.

Results:

1. When rats were fed the normal (1%) calcium diet, less than 1% of theadministered dose of ¹⁴C from oxalate was recovered in expired air (ascarbon dioxide produced from ¹⁴C oxalate in the intestine, absorbed intoblood and then expired) however in all cases more of the ¹⁴C wasrecovered during the period when rats were fed Oxalobacter cells (FIG.1a) This is in contrast to results obtained when the diet was low incalcium (0.02%) when more than 50% of the ¹⁴C from oxalate was recoveredas carbon dioxide in expired air during the experimental period whenrats were fed Oxalobacter cells (FIG. 1b). These results are strikinglydifferent from the very low quantities of ¹⁴C (less than 5%) recoveredduring the control period (before the feeding of Oxalobacter cells).Thus feeding Oxalobacter formigenes cells to rats markedly increased theamount of dietary oxalate that was degraded in the intestinal tract.

2. Feeding Oxalobacter cells also decreased the amount of ¹⁴C-oxalatethat was excreted in urine. Values for a 4 day collections during boththe control and experimental periods and for a single day in each ofthese periods are shown in FIGS. 2a and 2 b respectively. Quantities ofoxalate recovered in rat feces were also lower during the experimentalperiod (when Oxalobacter cells were fed) than was found for the controlperiod (FIG. 2c).

Most laboratory rats do not carry Oxalobacter in their intestinal tracts(they are not colonized). The present results show that purposefuladministration of these oxalate-degrading bacteria to rats causes alarge portion of the dietary oxalate to be degraded and thatconsequently less of the oxalate from the diet is excreted in urine.

The effects of dietary calcium on oxalate degradation are marked.Calcium complexes with oxalate so that its solubility and availabilityfor attack by Oxalobacter is limited and the amount that is degradedwhen rats are fed a high calcium diet is much less than amounts degradedwhen calcium in the diet is low.

EXAMPLE 5

Effect of Feeding O. formigenes on Urinary Oxalate Excretion in Pigs

Pigs are naturally colonized with Oxalobacter. Decolonization wasachieved in experimental pigs by antibiotic supplementation of the diet.Pigs were fed Oxalobacter in culture broth, which they readily consumed.The pigs were fed a soybean/corn based feed supplemented with 1300 mgoxalate/kg. The basal diet contained 680 mg oxalate/kg. Results areshown in FIGS. 3a-c for three individual pigs.

In all the three pigs urinary oxalate was dramatically decreased duringthe consumption of Oxalaobeter. The level of excretion of oxalate inthese pigs decreased to a minimum of approximately 6 mg/g creatinine inall three pigs. This is to be compared with a level of 8-10 mg/gcreatinine that has been observed in humans taking oxalate-free formuladiets. This level is equated to the endogenous synthesis in humans asthe dietary load has been eliminated. It appears that this levelreflects endogenous synthesis in pigs and that the intestinal absorptionhas been eliminated by Oxalobacter treatment. Furthermore, these resultsindicate that the ingested Oxalobacter were able to remove both thecrystalline oxalate added and the food-borne oxalate that wasbioavailable.

In this experiment each pig was fed 1.0 g of cell paste with the morningmeal. At O.D.₆₀₀ of 0.6, viable cell count is 2.1×10⁸ cells/ml, whichextrapolates to 2.1×10¹³ cells per 100 L. The 100 L fermenter runprovides us on the average 50-60 gm wet wt of cells. Therefore, 1 gm wetwt of cells is about 3.5×10¹¹ viable cells.

The dose of 3.5×10¹¹ viable cells as indicated above could eliminateintestinal absorption of about 2.0 gm of oxalate present per kg diet(1300 mg added oxalate+680 mg present in the diet). The animals consumed1 kg diet per meal.

The body weight of the pigs is about 200 lbs. and the digestive systemof the pigs is believed to be very close to that of humans. In humansthe average daily consumption of oxalate is about 100-400 mg dependingon the diet composition which is also split into three meals/day,therefore on an average a daily dose of 10⁸ to 10¹⁰ viable cells wouldbe sufficient to prevent the dietary absorption of oxalate.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

What is claimed is:
 1. A method for reducing oxalate concentrations inan animal wherein said method comprises administering a compositioncomprising a material selected from the group consisting of anoxalate-reducing effective amount of oxalate-degrading microbes andoxalate-degrading enzymes.
 2. The method, according to claim 1, whereinsaid method comprises administration of oxalate-degrading enzymes. 3.The method, according to claim 2, wherein said oxalate-degrading enzymesare derived from bacteria.
 4. The method, according to claim 3, whereinsaid oxalate-degrading enzymes are derived from bacteria of the groupconsisting of Clostridium, Pseudomonas, and oxalobacter.
 5. The method,according to claim 2, wherein said enzymes are produced recombinantly.6. The method, according to claim 5, wherein said enzymes are producedrecombinantly in Escherichia coli.
 7. The method, according to claim 2,which comprises administering formyl-CoA transferase and oxalyl-CoAdecarboxylase.
 8. The method, according to claim 7, wherein said enzymesare produced recombinantly.
 9. The method, according to claim 2, whereinsaid oxalate-degrading enzymes are expressed in plants which have beentransformed with polynucleotides encoding said oxalate-degradingenzymes.
 10. The method, according to claim 1, wherein said methodcomprises administration of oxalate-degrading microbes.
 11. The method,according to claim 10, wherein said oxalate-degrading microbes have beentransformed with polynucleotides which encode said oxalate-degradingenzymes.
 12. The method, according to claim 2, which further comprisesadministering an additional factor selected from the group consisting ofoxalyl CoA, MgCl₂ and TPP.
 13. The method, according to claim 10, whichcomprises administering whole viable oxalate-degrading microbes.
 14. Themethod, according to claim 13, wherein said microbes are Oxalobacterformigenes.
 15. The method, according to claim 13, wherein said microbesare selected from the group consisting of Clostridium and Pseudomonas.16. The method, according to claim 13, wherein said microbes colonizethe intestines.
 17. The method, according to claim 1, which is used totreat a patient whose intestines have insufficient numbers ofoxalate-degrading bacteria.
 18. The method, according to claim 17, whichis used to treat a patient whose natural intestinal bacteria have beendepleted due to treatment with antibiotics.
 19. The method, according toclaim 1, which is used to treat a domesticated animal, said animalhaving deficient numbers of oxalate-degrading bacteria.
 20. The method,according to claim 19, wherein said domesticated animal is selected fromthe group consisting of dogs, cats, rabbits, ferrets, guinea pigs,hamsters and gerbils.
 21. The method, according to claim 19, whereinsaid domesticated animal is an agricultural animal.
 22. The method,according to claim 21, wherein said agricultural animal is selected fromthe group consisting of horses, cows and pigs.
 23. The method, accordingto claim 19, which is used treat a domesticated animal, said animal'snatural intestinal bacteria having been depleted due to treatment withantibiotics.
 24. The method, according to claim 1, wherein said microbeor said enzyme is formulated to reduce inactivation in the stomach. 25.The method, according to claim 24, wherein said formulation comprises acoating which dissolves preferentially in the small intestine comparedto the stomach.